Central to photoreceptor cell biology is an understanding of the sorting and transport of membrane proteins, both integral and peripherally membrane-associated, within the inner segment. Integral membrane proteins are synthesized by ER-associated ribosomes and exported to the Golgi apparatus. Prenylated or acylated peripheral membrane proteins are synthesized in the cytosol and become ER-associated. Vesicles emerge from the trans-Golgi network (TGN) and transport to the base of the cilium where fusion with the cell membrane occurs. Finally, cargo is assembled for intraflagellar transport. Based on our previous results with guanylate cyclase knockouts, we postulate that some peripheral membrane-associated proteins (PMPs) of the phototransduction machinery cotransport with GC-containing vesicles. In GC1/GC2 double knockout rods, PDE6 failed to transport whereas transducin (T) and opsin kinase (GRK1) were unaffected. In GC1-/- cones, the entire phototransduction machinery was absent from the COS suggesting that formation of a large cargo requires GC1, itself an integral membrane protein. Based on deletion of a prenyl binding protein, PrBP/d (Pde6d), we discovered that transport of geranylgeranylated PDE6C and farnesylated GRK1 to cone outer segments was blocked while rod PDE6 and rod T transported as expected. Aim 1 of this proposal will address the identification and functional characterization of novel putative prenyl/acyl binding proteins (UNC119 and UNC119B) by gene knockdowns and knockouts. Aim 2 will examine the role of GC1 in post-Golgi transport of cone membrane proteins, emphasizing the importance of the GC1 extracellular domain and GC activity, as well as the role of kinesin-II in intraflagellar transport (IFT). Aim 3 will elucidate the roles of Ca2+-binding proteins, centrin-1 and centrin-2, in intraflagellar transport by rod- and cone- specific knockouts of their genes. These studies will collectively expand our knowledge regarding the protein functions and interactions that participate in photoreceptor maintenance and renewal. PUBLIC HEALTH RELEVANCE: Photoreceptor outer segments are renewed every ten days. Daily renewal of ~10% (about 100 disks) of the outer segment disk stack requires a continuous high rate of biosynthesis, reliable transport, and targeting pathways to replace outer segment proteins. A central question in photoreceptor cell biology concerns post-biosynthetic transport of membrane-associated proteins and the mechanisms of targeting to the outer segments for disk assembly - pathways that are essential for cell maintenance and survival. Otherwise stated, "How does the highly polarized photoreceptor build an outer segment?" The proposed research examines aspects of integral membrane and peripheral membrane protein transport from photoreceptor inner segment (site of biosynthesis) to the outer segment (site of phototransduction). In aim 1, the role of prenyl- or acyl binding proteins will be investigated. In aim 2, we will explore the roles of guanylate cyclase 1 and kinesin-II, a molecular motor involved in intraflagellar transport. Finally in aim 3, the consequences of photoreceptor-specific deletions of centrin- 1 will be studied. Transport of membrane proteins, particularly post-Golgi transport and intraflagellar transport through the cilium, are complex and insufficiently understood in disease etiology.